Objectives: This in vitro study aimed to evaluate and compare the effect of two different bioactive glasses, a hydroxyapatite-containing, fluoride-free toothpaste (HTP) and a fluoride toothpaste (FTP) on the remineralization behavior of initial caries lesions. Materials and Methods: A total of 100 bovine enamel samples were randomly allocated to five groups of 20 samples each: NC = negative control group (artificial saliva); HTP = HTP group (Karex); FTP = FTP group (Elmex caries protection, 1,400 ppm); FTP + BG_nano = FTP followed by Actimins bioactive glass; FTP + BG_amorph = FTP followed by Schott bioactive glass. Radiographic documentation (advanced transversal microradiography; aTMR) was applied before and after all samples were exposed to a demineralizing gel for 10 days. Over a period of 28 days, samples were covered twice a day (every 12 h) with a toothpaste slurry of the respective test group or with artificial saliva in NC for 60 s and brushed with 15 brushing strokes. Samples in FTP + BG_nano and FTP + BG_amorph were additionally treated with the respective bioactive glass slurry for 30 s after brushing with the FTP. In the meantime, all samples were stored in artificial saliva. After 28 days, the structure of all samples was assessed again using aTMR and compared to the values measured after demineralization. The statistical evaluation of the integrated mineral loss was performed using Kruskal-Wallis test followed by a post hoc Conover test. Results: The FTP revealed the significantly highest increase of mineral content while the HTP showed the significantly lowest remineralization. Compared to artificial saliva, the use of the HTP or the combined application of FTP followed by bioactive glasses (FTP + BG_nano and FTP + BG_amorph) showed no significant remineralization. Conclusion: Under remineralizing in vitro conditions, brushing with 1,400 ppm FTP induced significantly more remineralization compared to storage in artificial saliva. The additional administration of both bioactive glasses as well as the substitutional brushing with an HTP resulted in significantly less remineralization compared to brushing with 1,400 ppm FTP.